Recapitulating protein vectorial appearance enhances folding to the native structure in vitro

In the cell, protein folding starts vectorially, in a specific direction determined by protein synthesis (N- to C-terminus) or secretion (either N- to C- or C- to N-terminus, depending on the secretion mechanism). In contrast, during a typical in vitro protein refolding experiment, the abrupt removal of a chemical denaturant enables refolding to begin with interactions formed between any portions of a full-length polypeptide chain. The vectorial appearance of the polypeptide chain enables proteins to begin folding by exploring a simple energy landscape that increases in size and complexity as the amino acid chain lengthens. However, currently little is known regarding the impact of vectorial folding on protein folding kinetics and/or aggregation avoidance, in part because no simple method exists to test the impact of vectorial appearance on protein folding mechanisms in vitro. In this study, we adapted the ring-shaped AAA+ translocase ClpX to create an experimental platform with which to study vectorial protein folding in vitro. Crucially, translocation through the central nanometer-sized pore of ClpX enables us to recapitulate vectorial appearance of polypeptide chains from either N- to C- or C- to N-terminus. We used this approach to study the impact of vectorial folding of three diverse fluorescent proteins. Whereas these proteins are highly prone to misfolding and aggregation under traditional in vitro refolding conditions, initiating folding by translocation through ClpX led to robust folding to the native structure. We are currently adapting this assay to study the impact of vectorial chain appearance on protein folding mechanisms.